METTL14-mediated Lnc-LSG1 m6A modification inhibits clear cell renal cell carcinoma metastasis via regulating ESRP2 ubiquitination

Clear cell renal cell carcinoma (ccRCC) is the most lethal urological cancer and is characterized by a high rate of metastasis and relapse. N6-Methyladenosine (m6A) is implicated in various stages of cancer development. However, a thorough understanding of m6A-modified lncRNAs in ccRCC is lacking. The results showed that METTL14 had decreased expression in ccRCC tissues. In addition, the expression of METTL14 was negatively correlated to the prognosis, stage, and ccRCC tumor grade. The silencing of METTL14 was shown to significantly increase metastasis in vitro and in vivo. High-throughput methylated RNA immunoprecipitation sequencing (MeRIP-seq) showed that the m6A levels of Lnc-LSG1 could be regulated by METTL14. Lnc-LSG1 can directly bind to ESRP2 protein and promote ESRP2 degradation via facilitating ESRP2 ubiquitination. However, m6A modification on Lnc-LSG1 can block the interaction between Lnc-LSG1 and ESRP2 via the m6A reader, YTHDC1. Taken together, our findings unraveled the novel mechanism of METTL14 inhibiting ccRCC progression, and explored the correlation between m6A and lncRNA in ccRCC for the first time.


INTRODUCTION
Renal cell carcinoma (RCC) accounts for 4% of all malignancies and is the most lethal urological cancer in the United States. 1 Approximately 90% of RCCs are clear cell RCC (ccRCC) which is characterized by a high rate of metastasis and relapse. Nephrectomy is considered as the gold standard for RCC treatment. However, about 30% of ccRCC patients present with metastasis at the initial diagnosis. In addition, up to one-third of patients with early-stage ccRCC develop metastasis following nephrectomy, thus poor prognosis. 2 Therefore, exploring the mechanisms underlying metastasis and identifying new biomarkers for predicting ccRCC progression may give insights into the development of highly efficacious therapies.
N6-Methyladenosine (m 6 A) is the most prevalent mRNA modification in eukaryotic cells. 3 It is a dynamic and reversible process involved in distinct mRNA metabolism phases, including splicing, 4 translation, 5 nuclear export, 6 and stability. 7 It is catalyzed by an m 6 A methyltransferase complex composed of methyltransferase 3 (METTL3), methyltransferase 14 (METTL14), and WT1-associated protein (WTAP). The m 6 A methylation can be reversed by m 6 A demethylases, including fat mass and obesity-associated protein (FTO) and alkB homolog 5 (ALKBH5). The methylation is detected by m 6 A "reader" proteins, which include YTH domain proteins, including YTH m 6 A RNA binding protein 1-3 (YTHDF1-3) and YTH domain containing 1-2 (YTHDC1-2), heterogeneous nuclear ribonucleoproteins, and insulin-like growth factor-2 mRNAbinding proteins. 8 Recently, numerous studies have implicated the dysregulation of m 6 A modification in several pathological processes. 9,10 A previous study evaluated the prognostic value of m 6 A readers and writers in ccRCC by analyzing RNA sequencing and copy number variations (CNVs) data from The Cancer Genome Atlas (TCGA) database obtained from 528 ccRCC patients. The results revealed that CNVs in the m 6 A readers and writers were associated with poorer overall survival (OS) and disease-free survival (DFS). 11 However, the detailed molecular mechanisms through which m 6 A modification regulates ccRCC progression have not been elucidated.
Previous studies mainly focused on how the m 6 A modifications determined the fate of mRNAs. Recent accumulating evidence has shed light on the correlation between m 6 A methylation and noncoding RNAs (ncRNAs). 12,13 An m 6 A mapping study reported that m 6 A methylation was also extensively present in long noncoding RNAs (lncRNAs). 14 Furthermore, m 6 A was shown to influence cancer progression by regulating the metabolism and functions of lncRNAs. 7,15,16 This suggests that m 6 A-modified lncRNAs may play a crucial role in cancer. To date, no study has explored the role of m 6 A modification and lncRNAs in ccRCC.
In this study, we found that METTL14 functions as a tumor suppressor in ccRCC metastasis using patients' samples from our own cohort and a ccRCC tissue microarray, as well as in vitro and in vivo experiments. By performing high-throughput methylated RNA immunoprecipitation sequencing (MeRIP-seq) and Transwell assay, we identified Lnc-LSG1 as the downstream target of anti-metastatic function of METTL14. Further experiments demonstrated that m 6 A on Lnc-LSG1 blocked the interaction between Lnc-LSG1 and epithelial splicing regulatory protein 2 (ESRP2) protein via YTHDC1, thus protecting ESRP2 from ubiquitinated degradation. Our findings expand the understanding of the role and underlying mechanisms of METTL14-mediated lncRNA m 6 A modification in ccRCC progression and prognosis, and provide a new insight into ccRCC metastasis from the aspect of the epigenetic modification of noncoding RNAs.

Decreased METTL14 expression was associated with poor prognosis
The total m 6 A levels in nine pairs of ccRCC tumor tissues and the paired peritumor tissues were determined using the m 6 A RNA methylation quantification kit to investigate the potential role of m 6 A modification in ccRCC. The results revealed that the m 6 A levels were significantly decreased in the ccRCC tumor tissues ( Figure 1A). The decreased m 6 A levels in the ccRCC tissues were probably due to a dysregulation of the writers and erasers since the m 6 A modification is mainly catalyzed by m 6 A writers and erasers.
The expression profiles of key m 6 A writers (METTL3, METTL14, and WTAP) and erasers (ALKBH5 and FTO) in 50 pairs of ccRCC tissues obtained from patients who had undergone nephrectomy at Sir Run Run Shaw Hospital (SRRSH) was determined. The qRT-PCR assay showed that METTL14, an essential component of the m 6 A methyltransferase complex, 17 was significantly downregulated in ccRCC. However, there were no significant differences in the expression of METTL3, WTAP, ALKBH5, and FTO ( Figure 1B). Moreover, protein levels of METTL14 were significantly decreased in the ccRCC tissues than in their adjacent normal tissues ( Figure 1C). Subsequently, results showing decreased METTL14 protein expression were validated in 40 ccRCC samples obtained from SRRSH cohort and in a tissue microarray constructed from 79 ccRCC samples based on immunohistochemistry (IHC) staining ( Figures 1D and 1E). The results revealed that METTL14 was significantly decreased in the ccRCC tissues.
Furthermore, patients with low expression of METTL14 showed poorer OS, suggesting that METTL14 plays a tumor-suppressor role in ccRCC ( Figure 1F). These results were consistent with the ccRCC data mining results from Database: UALCAN (http:// ualcan.path.uab.edu/), which showed that METTL14 had decreased expression in ccRCC tissues ( Figure S1A). Moreover, levels of METTL14 were negatively correlated to the tumor stage, grade, and nodal metastasis status ( Figures S1B and S1C). Finally, the Kaplan-Meier survival analysis of data obtained from Database: GEPIA (http://gepia.cancer-pku.cn/) also showed that decreased expression of METTL14 was associated with poor OS and DFS (Figures S1D and S1E). Taken together, these data suggest that METTL14 might be involved in ccRCC progression and is a potential prognostic indicator in ccRCC patients.

METTL14 inhibited metastasis in ccRCC via m 6 A modification
We generated METTL14-overexpressing and knockdown cell models in 786-O, Caki-1, and OSRC-2 cells to evaluate whether METTL14 was negatively correlated to ccRCC progression. The overexpression or knockdown of METTL14 was confirmed by determining the mRNA and protein levels (Figures 2A, 2B, and S2A). The proliferative and metastatic abilities were assessed by using CCK8 and Transwell assays, respectively. Results of the CCK8 assay showed that METTL14 did not affect cell proliferation ( Figure S2B). However, METTL14 knockdown significantly increased the migration and invasion ability of ccRCC cells. In contrast, overexpression of METTL14 significantly decreased the migration and invasion ability of the ccRCC cells (Figures 2C and S2C). These results suggest that METTL14 is a negative regulator in ccRCC metastasis. Furthermore, the wound healing assay showed similar results ( Figures 2D and S2D).
Moreover, we constructed plasmids expressing wild-type METTL14 (METTL14-WT) and mutant METTL14 (METTL14-R298P; R298 is critical for the target recognition of the methyltransferase complex 17,18 ) to determine whether the effect of METTL14 on metastasis was dependent on its ability to recognize m 6 A targets. As shown in Figure 2E, the ectopic expression of METTL14-WT, but not METTL14-R298P, reversed the increased migration and invasion induced by METTL14 silencing. These observations suggested that METTL14 inhibited in vitro migration and invasion in ccRCC in an m 6 A-dependent manner.
Furthermore, we established stable METTL14 knockdown in luciferase-expressing OSRC-2 cells to investigate the anti-metastatic effect of METTL14 in ccRCC in vivo. The control and METTL14 knockdown cells were orthotopically injected into the renal capsule of BALB/c nude mice. The metastatic ability was then determined using a bioluminescence imaging system after 6 weeks. The METTL14 knockdown group showed increased cancer metastasis ( Figure 2F) and a higher metastatic ratio than the control group ( Figure 2G). Subsequently, we established a ccRCC lung metastasis model by injecting METTL14 knockdown and corresponding control cells into the tail vein of BALB/c nude mice. The silencing of METTL14 was shown to significantly promote lung metastases, as shown by increased bioluminescent signals in the lungs ( Figure 2H). Moreover, hematoxylin and eosin (H&E) staining showed a higher number of metastatic tumor nodules in the lungs of the METTL14 knockdown group than in the control group ( Figure 2I). We also detected the effect of METTL14 on tumor proliferation. The results showed that the volume of the tumors was nearly the same in the shMETTL14-1 and control groups ( Figure S2E), consistent with the CCK8 results.
Lnc-LSG1 was identified as a downstream target of METTL14 METTL14 is a component of the m 6 A methyltransferase. Knockdown of METTL14 leads to decreased m 6 A levels. To date, no studies have investigated m 6 A methylation of lncRNAs in ccRCC. This study investigated whether lncRNAs m 6 A methylation mediated the anti-metastatic effect of METTL14. Therefore, we    Table S6). Among these, we selected Lnc-IL17B-2, Lnc-ZNF121-1, Lnc-LSG1-4:2, and Lnc-ENPP1-5 for further investigation (logFC < À1, p < 0.05) ( Figure 3A). Results of the m 6 A levels of the four lncRNAs after METTL14 knockdown were validated using MeRIP and qRT-PCR assay. As shown in Figure 3B, only Lnc-ZNF121-1 and Lnc-LSG1-4:2 exhibited decreased m 6 A levels after METTL14 knockdown in the 786-O and Caki-1 cells, with Lnc-LSG1-4:2 (Lnc-LSG1) exhibiting the highest decrease in m 6 A levels. The Integrative Genomics Viewer plots also showed a decreased m 6 A peak at the Lnc-LSG1 transcripts ( Figure 3C). The 5 0 and 3 0 rapid amplification of cDNA ends analysis showed that Lnc-LSG1 had a full length of 980 nt ( Figure S2F). Furthermore, we compared the expression of Lnc-LSG1 with several highly expressed lncRNAs in ccRCC to investigate the abundance of Lnc-LSG1. [19][20][21] The qRT-PCR assay revealed that the expression of Lnc-LSG1 was higher than MALAT1 in the 786-O cells, and UCA1 in the 786-O and OSRC-2 cells. These results suggested a high abundance of Lnc-LSG1 in the ccRCC cells ( Figure S2G). Bioinformatics analysis using the online database Coding Potential Calculator 2.0 (CPC 2.0, http://cpc2.gao-lab.org/index.php) showed that Lnc-LSG1 could not code for proteins 22 ( Figure S2H). The Transwell assay showed that knockdown of Lnc-LSG1 decreased the migration and invasion ability of ccRCC cells. In contrast, overexpression of Lnc-LSG1 increased the migration and invasion ability of ccRCC cells, suggesting that Lnc-LSG1 could be a metastasis-related lncRNA ( Figures  3D, S2I, and S2J). Moreover, knockdown of Lnc-LSG1 effectively reversed the increased metastatic ability of METTL14 knockdown cells ( Figure 3E). We also investigated the effect of Lnc-LSG1 on cell proliferation using in vitro and in vivo assays. The results showed that Lnc-LSG1 did not influence the proliferation of ccRCC cells ( Figures S2K and S2L). Therefore, Lnc-LSG1 was considered as a candidate substrate of METTL14 for further investigation.

Lnc-LSG1 regulates ccRCC metastasis via binding to ESRP2 protein and repressing ESRP2 stability
We explored the functional mechanisms by which Lnc-LSG1 increased ccRCC metastasis. The qRT-PCR and fluorescence in situ hybridization (FISH) analysis of the fractionated cytoplasmic and nuclear RNA obtained from 786-O cells showed that Lnc-LSG1 was expressed in the nucleus and the cytoplasm, and mainly prevalent in the cytoplasm ( Figures 4A and 4B). LncRNA can activate the transcription of nearby genes in cis by promoting chromatin looping from transcriptional enhancers. 23,24 Therefore, we investigated the effect of Lnc-LSG1 on TMEM44, the neighboring protein-coding gene ( Figure S3A). There was no significant difference in the expression level of TMEM44 following Lnc-LSG1 knockdown or overexpression ( Figure S3B). This suggests that the biological functions of Lnc-LSG1 were not related to the cis-regulatory function.
LncRNAs are associated with a plethora of cellular functions, most of which require interaction with one or more proteins. Currently, available information points to an intricate network of protein-lncRNA interactions, whose dysregulation is associated with pathological states. 25 The online Database: catRAPID (http://s.tartaglialab.com/ page/catrapid_group) 26 predicted potential proteins that may interact with Lnc-LSG1. Epithelial-specific splicing regulator (ESRP2) 27 was identified as having the highest probability of interacting with Lnc-LSG1 ( Figure 4C). The Lnc-LSG1 pull-down and RIP assays with an anti-ESRP2 antibody were used to verify direct binding of Lnc-LSG1 to ESRP2 ( Figures 4D and 4E). Furthermore, we constructed a series of truncated Lnc-LSG1 and ESRP2 to determine the exact regions of interaction between Lnc-LSG1 and ESRP2. The Lnc-LSG1 nucleotides (0-300) were shown to be essential in the binding of ESRP2. Moreover, the RNA Recognition Motif 3 of ESRP2 was required for binding Lnc-LSG1 ( Figures 4F and 4G). These results confirmed that Lnc-LSG1 is a binding partner of ESRP2.
A previous study reported that ESRP2 knockdown enhanced cell migration in ccRCC. 28 Therefore, we hypothesized that Lnc-LSG1 promoted ccRCC metastasis via ESRP2. Results of the western blotting analysis demonstrated that overexpression of Lnc-LSG1 significantly decreased protein levels of ESRP2. In contrast, Lnc-LSG1 knockdown increased ESRP2 protein levels ( Figures 4H and S3C). The qRT-PCR analysis showed that mRNA levels of ESRP2 were not affected by Lnc-LSG1 ( Figure S3D). Furthermore, we hypothesized that Lnc-LSG1 promoted degradation of ESRP2 protein based on the fact that lncRNAs modulate protein stability via direct interaction. 24,29 Therefore, we treated ccRCC cells with a protein translation inhibitor, cycloheximide (CHX), to block ESRP2 synthesis. After that, we detected the ESRP2 protein levels using western blotting. The results showed that overexpression of Lnc-LSG1 shortened the half-life of the ESRP2 protein ( Figure 4I). Furthermore, the ESRP2 protein levels were significantly increased after treatment with MG132 (a proteasome inhibitor) compared with chloroquine treatment (an autophagy inhibitor) ( Figure 4J). These results imply that Lnc-LSG1-induced ESRP2 degradation may be dependent on the ubiquitin-proteasome pathway. Accordingly, overexpression of Lnc-LSG1 significantly increased the ESRP2 ubiquitination levels ( Figure 4K). The transwell assay confirmed that ESRP2 knockdown promoted ccRCC migration and invasion ( Figures 4L, S3E, and S3F). In addition, impaired metastatic ability in Lnc-LSG1-silenced cells could be restored by knocking down ESRP2 ( Figures 4M and S3G), suggesting that ESRP2 was a potential downstream target of Lnc-LSG1. Taken together, these results reveal that Lnc-LSG1 promotes ccRCC metastasis by directly binding to the ESRP2 protein, thus inhibiting its expression through the ubiquitin-proteasome pathway.

METTL14 increases ESRP2 protein stability via Lnc-LSG1
Furthermore, we sought to investigate whether ESRP2 was a downstream target of METTL14 in inhibiting ccRCC metastasis. The anti-m 6 A RIP assay found that ESRP2 mRNA had no m 6 A modification. In addition, METTL14 did not regulate ESRP2 m 6 A levels. Based on these findings, we ruled out the possibility of METTL14 directly www.moleculartherapy.org  modifying ESRP2 via m 6 A ( Figure 5A). The qRT-PCR assay showed that METTL14 did not affect the mRNA expression of ESRP2 (Figure S4A). Western blotting analysis indicated that the ectopic expres- sion of METTL14-WT, but not METTL14-R298P, increased ESRP2 protein levels, suggesting that METTL14 promotes ESRP2 protein expression through regulating m 6 A methylation ( Figure 5B). Conversely, ESRP2 protein expression was inhibited in shMETTL14 cell lines (Figure 5B). Moreover, IHC staining showed decreased expression of ESRP2 in orthotopic tumors injected with METTL14 knockdown cells ( Figure 5C). We also used 40 ccRCC samples in Figure 1D for IHC analysis and calculated the ESRP2 protein IHC score. Then, Pearson's correlation analysis showed that the METTL14 protein expression was positively correlated with ESRP2 protein expression (r = 0.401, p = 0.010; Figure 5D).
In addition, METTL14 was shown to extend the half-life of ESRP2 protein ( Figure 5E) and reduce its ubiquitination level ( Figure 5F). However, shMETTL14 showed opposite effects (Figures 5E and 5F). Importantly, ESRP2 reversed the increased metastatic ability induced by the knockdown of METTL14 in ccRCC cells (Figures 5G and S4B). These findings suggest that METTL14 inhibits ccRCC metastasis by decreasing ESRP2 ubiquitination and increasing ESRP2 protein levels.
Furthermore, we transfected the Lnc-LSG1 smart silencer into shMETTL14 cell lines to determine whether METTL14 regulated ESRP2 protein levels through Lnc-LSG1. Western blotting assay demonstrated that the METTL14 knockdown-induced downregulation of ESRP2 protein could be restored by knockdown of Lnc-LSG1 ( Figure 5H). Furthermore, knockdown of Lnc-LSG1 decreased the ubiquitination of ESRP2 induced by shMETTL14 ( Figure 5I). In summary, these results indicate that METTL14 increases ESRP2 protein stability via Lnc-LSG1.

METTL14 inhibits the interaction between ESRP2 and Lnc-LSG1 through YTHDC1
Furthermore, we investigated the detailed mechanism of how METTL14 regulated ESRP2 protein degradation through Lnc-LSG1. METTL14 and Lnc-LSG1 show opposing effects on the ESRP2 protein. In addition, METTL14 regulates m 6 A methylation of Lnc-LSG1. Therefore, we hypothesized that METTL14 could inhibit the function of Lnc-LSG1 in an m 6 A-dependent manner. The qRT-PCR assay showed that METTL14 did not regulate the expression of Lnc-LSG1 ( Figure S4C). Most studies showed that m 6 A methylation regulates lncRNA function by modulating lncRNA stability. 30,31 However, other authors reported that m 6 A methylation might also be directly involved in lncRNA-RNA and lncRNA-protein interactions. For example, a recent study by Yoneda et al. reported that m 6 A modification in lncRNA pcnRNA-D abolished the direct binding of pncRNA-D to the protein TLS through an m 6 A reader YTHDC1. 32 The Lnc-LSG1 facilitates ESRP2 ubiquitination through direct binding. In addition, METTL14 reduces ESRP2 ubiquitination. Based on these facts, we hypothesized that METTL14 inhibited the binding of ESRP2 and Lnc-LSG1 by promoting YTHDC1 binding to the m 6 A sites on Lnc-LSG1. This is similar to the effect of m 6 A-YTHDC1 on the interaction between pcnRNA-D and TLS protein. 32 The anti-YTHDC1 RIP assay showed that YTHDC1 had significantly increased binding to the Lnc-LSG1 than the IgG control ( Figure 6A). Furthermore, anti-YTHDC1 RIP assay showed that the interaction between YTHDC1 and Lnc-LSG1 could be regulated by METTL14 ( Figure 6B), and qRT-PCR assay showed that YTHDC1 did not affect mRNA expression of ESRP2 ( Figure S4D), suggesting that YTHDC1 was the m 6 A reader of Lnc-LSG1 m 6 A methylation. The anti-ESRP2 RIP assay showed increased ESRP2/Lnc-LSG1 interaction in shMETT14 cells and decreased interactions in the OE-METTL14 cells (Figures 6C and 6D). However, METTL14-R298P had no effect on ESRP2/Lnc-LSG1 interaction ( Figure 6E), which suggested that the inhibitory function of METTL14 on Lnc-LSG1/ESRP2 interaction is mediated by m 6 A. Moreover, siYTHDC1 enhanced the binding of Lnc-LSG1 to ESRP2 protein and abrogated the inhibited ESRP2/ Lnc-LSG1 interaction induced by METTL14 overexpression (Figure 6D). These results provide evidence that METTL14 regulates the binding between ESRP2 and Lnc-LSG1 via YTHDC1. M 6 A modification tends to occur in an RRACH (R: A or G; H: A, C, or U) consensus sequence, 33 especially the GGACU. [34][35][36] To identify the adenosine residue(s) on Lnc-LSG1 responsible for YTHDC1 binding, we searched for the RRACH motif in nucleotides 0 to 300 of Lnc-LSG1, required for the ESRP2/Lnc-LSG1 interaction ( Figure 4F). The results showed four RRACH motifs and only one GGACU sequence in nucleotides 0 to 300 ( Figure 6F). We then generated a 25 nt biotinylated RNA probe around GGACU with or without m 6 A methylation (probe m 6 A and probe A) to investigate the function of m 6 A methylation in the GGACU sequence ( Figure 6G). Subsequently, we conducted an RNA pull-down assay using the two probes. Furthermore, we determined the precipitated protein levels of YTHDC1 and ESRP2. The results showed that YTHDC1 could bind to probe m 6 A, but not to the non-methylated probe A (Figure 6H). On the other hand, more ESRP2 protein was shown to bind to probe A than probe m 6 A ( Figure 6H), suggesting that m 6 A inhibits ESRP2 binding to Lnc-LSG1. These results confirm that YTHDC1 competitively inhibits ESRP2 binding to Lnc-LSG1 in an m 6 A-dependent manner.
Furthermore, we investigated the effect of YTHDC1 on ESRP2 expression. As shown in Figure 6I, ESRP2 protein expression can be upregulated by overexpression of YTHDC1 and downregulated by knockdown of YTHDC1. However, ESRP2 mRNA expression was not regulated by YTHDC1 ( Figure S4D). In addition, YTHDC1 could partially reverse the inhibitory effect of Lnc-LSG1 on ESRP2 protein without altering Lnc-LSG1 expression (Figures 6J and S4D). Moreover, siYTHDC1 can block the effect of METTL14 overexpression on ESRP2 expression and ubiquitin modification ( Figures 6K  and 6L). Taken together, these results suggest that METTL14 can inhibit ESRP2 and Lnc-LSG1 interaction by increased binding of YTHDC1 to the m 6 A site in Lnc-LSG1. DISCUSSION m 6 A methylation is the most prevalent post-transcriptional RNA modification in eukaryotic cells. It is reversible and catalyzed by corresponding enzymes, namely "writers," "erasers," and "readers." Numerous studies have explored the dysregulation and specific functions of m 6 A enzymes in various diseases and malignancies. 37,38 In this study, bioinformatics analysis was used to determine the role of m 6 A modification in ccRCC progression. Using RNA sequencing and CNV data of ccRCC patients from TCGA database, Zhou et al. reported that genetic alterations of m 6 A regulators in ccRCC were associated with decreased m 6 A levels and poorer OS and DFS. 11 Zhong et al. clustered ccRCC patients into three m 6 A modification patterns with distinct immune landscapes and prognoses. They reported that the m 6 A gene signature was an independent prognostic factor for ccRCC. 39 In addition, Zhou et al. 11 and Gong et al. 40 found that METTL14 was downregulated in the TCGA KIRC cohort and was negatively associated with the ccRCC stage and OS. However, these findings were based on data obtained from online databases. Moreover, research based on in vitro and in vivo experiments is limited. Therefore, little information is currently available about the biological functions and molecular mechanisms of METTL14 in ccRCC progression.
www.moleculartherapy.org In this study, the decreased expression of METTL14 was verified in samples obtained from ccRCC patients in the SRRSH cohort and tissue microarrays. Furthermore, the results revealed that low METTL14 expression was correlated with poorer OS. In addition, METTL14 significantly inhibited the ccRCC migration and invasion in vitro and in vivo, suggesting that METTL14 has a tumor-suppressor role. M 6 A modification has also been identified in lncRNAs. Recently, several studies have shown that m 6 A modification in lncRNAs is implicated in various cancers. For instance, METTL3-and YTHDF3-mediated m 6 A methylation was shown to enhance the stability of MALAT1 in non-small cell lung cancer (NSCLC). MALAT1 functions as an endogenous RNA (ceRNA) sponging miR-1914-3p, thereby increasing expression of YAP, resulting in NSCLC metastasis and cisplatin resistance. 15 In this study, MeRIP-seq was used to explore the functions of m 6 A-modified lncRNAs in ccRCC for the first time. The results revealed that Lnc-LSG1 was a downstream target of METTL14 with an anti-metastastic effect on ccRCC cells. Furthermore, METTL14 was shown to inhibit binding between Lnc-LSG1 and ESRP2 protein by recruiting YTHDC1 to a GGACU motif on Lnc-LSG1, eventually increasing the stability of ESRP2 protein.
LncRNAs have been shown to regulate protein stability through RNA-protein interactions. 41,42 In this study, the RNA pull-down and RIP assays showed a direct protein interaction between ESRP2 and Lnc-LSG1. The results revealed that Lnc-LSG1 inhibited ESRP2 protein expression by shortening its half-life, suggesting that the interaction between Lnc-LSG1 and ESRP2 could regulate ESRP2 expression through protein degradation. Treatment with MG132 can significantly increase ESRP2 protein levels. Therefore, we investigated the functional significance of Lnc-LSG1 in the ubiquitination of ESRP2 protein. Lnc-LSG1 was shown to increase ESRP2 ubiquitination levels, implying that Lnc-LSG1 promotes ESRP2 degradation via the ubiquitination pathway. Epithelial splicing regulatory proteins (ESRP1 and ESRP2) are splicing regulators expressed in epithelial cells. According to Mizutani et al., ESRP2 expression, but not ESRP1, was maintained in ccRCC. Furthermore, they showed that the knockdown of ESRP2 promoted OSRC-2 migration. 28 Our data confirmed the function of ESRP2 in ccRCC cells.
This study shows that Lnc-LSG1 promoted ccRCC metastasis via binding the ESRP2 protein, further facilitating ubiquitination and degradation. According to Mizutani et al., Arkadia ubiquitinates lysine residues 27 (Lys27) on ESRP2, suppressing cell proliferation. However, the knockdown of Arkadia failed to show any effect on cell migration. Furthermore, ESRP2 ubiquitination by Arkadia did not induce ESRP2 degradation, 28 suggesting that ubiquitination on Lys27 by Arkadia was not responsible for ESRP2 protein degradation. Based on the fact that Lnc-LSG1-induced ESRP2 ubiquitination promotes ESRP2 degradation and enhances ccRCC migration, we hypothesized that Arkadia and Lys27 of ESRP2 do not mediate the Lnc-LSG1 function. Further studies are required to reveal the specific E3-ligase and lysine residues that mediate the ESRP2 polyubiquitination induced by Lnc-LSG1.
Zhao et al. reported a tissue-specific isoform switch of fibroblast growth factor receptor 2 (FGFR2) from FGFR2 IIIb (epithelial isoform) to FGFR2 IIIc (mesenchymal isoform) in nearly 90% of ccRCC patients. FGFR2 IIIc ccRCC were larger in size with worse clinical outcomes compared with FGFR2 IIIb ccRCC. 43 ESRP2 can induce a substantial switch from FGFR2 IIIc to FGFR2 IIIb. 27 Lnc-LSG1 was shown to increase FGFR2 IIIc expression and decrease FGFR2 IIIb expression using a qRT-PCR assay. In contrast, METTL14 was shown to decrease FGFR2 IIIc expression and increase FGFR2 IIIb expression ( Figure S4E). This is consistent with our finding that METTL14 promotes ESRP2 expression via Lnc-LSG1. Therefore, FGFR2 IIIb/IIIc was hypothesized to be a downstream target of ESRP2. However, the molecular mechanism of FGFR2 IIIb/IIIc involvement in ccRCC progression warrants further studies. YTHDC1 is responsible for RNA splicing by recognizing m 6 A methylation in RNAs. 44,45 Recently, Yoneda et al. proposed a competitive effect of YTHDC1 on RNA-protein interplay, and demonstrated that YTHDC1 can inhibit TLS protein binding to lncRNA pncRNA-D by m 6 A modification. 32 In this study, an RNA pull-down assay with m 6 A-modified and non-m 6 A-modified RNA probes showed that m 6 A methylation increases the binding of YTHDC1 to Lnc-LSG1. In contrast, m 6 A methylation decreases the binding of ESRP2 to Lnc-LSG1, implying that YTHDC1 competitively blocks the interaction between ESRP2 and Lnc-LSG1 by binding to m 6 A sites. In addition, the RIP assay showed that siYTHDC1 could disrupt the METTL14-induced inhibition on the interaction between Lnc-LSG1 and ESRP2. Furthermore, mutated METLL14 (METTL14-R298P) did not affect ESRP2 expression and ESRP2/ Lnc-LSG1 interaction. Knocking down YTHDC1 can partially reverse the effect of wild-type METTL14 on ESRP2 expression and ubiquitination. Taken together, these results reveal that YTHDC1, an m 6 A reader, is a mediator of METTL14 in inhibiting the interaction between Lnc-LSG1 and ESRP2.
In conclusion, we comprehensively elucidate the clinical relevance, functional roles, and detailed molecular mechanisms of METTL14 in ccRCC progression. For the first time, we provide insights into the function and mechanism of m 6 A-modified lncRNA in ccRCC and identify a "METTL14-YTHDC1-Lnc-LSG1" regulation axis in ccRCC progression. Thus, this study highlights the vital roles of METTL14 and lncRNA m 6 A modification in ccRCC development and may pave the way for developing novel biomarkers and therapies in ccRCC.

Human samples, cell lines, and antibodies
The ccRCC specimens and matched adjacent normal tissues were obtained from 50 patients who underwent curative surgical resections from 2018 to 2020 in the Department of Urology, SRRSH, Zhejiang University School of Medicine, Hangzhou, China. The protocols for www.moleculartherapy.org tissue sample collection were approved by the Ethics Committee of SRRSH (IRB no. 20180226-70). Informed consent was obtained from all patients. The clinical data of patients are provided in Tables S1 and S2. The tissue microarray was purchased from Shanghai Liao Ding Biotechnology (Shanghai, China). The clinical data of patients are provided in Table S3 The smart silencer targeting Lnc-LSG1, as well as siRNAs targeting ESRP2 and YTHDC1, were synthesized by RiboBio (Guangzhou, China); siRNA sequences are listed in Table S4. The smart silencer and siRNAs were transfected into cells using Lipofectamine RNAiMAX transfection reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's guidance. Ectopic expression plasmids of indicated genes were synthesized by GENECHEM (Shanghai, China) and transfected using Lipofectamine 3000 (Invitrogen). The METTL14-overexpressing and -knockdown lentivirus was designed, synthesized and collected by GENECHEM (Shanghai, China) and used to infect ccRCC cells with 8 mg/mL polybrene for 3 days. Stable infected cell lines were selected using puromycin (Selleck, Shanghai, China).

Bioinformatic analysis
The UALCAN (http://ualcan.path.uab.edu) online database was used to analyze the expression of METTL14 in normal and tumor tissues and in different ccRCC stages, grades, and metastasis status. GEPIA (http://gepia.cancer-pku.cn/) was used to investigate the prognostic role of METTL14. UALCAN and GEPIA tools deliver fast and customizable functionalities based on data from TCGA. They provide key interactive and customizable functions, including differential expression analysis, correlation analysis, and patient survival analysis. 46,47 The online tool catRAPID (http://s.tartaglialab.com/page/ catrapid_group) was used to predict the proteins that could interact with Lnc-LSG1. 26 The CPC 2.0 (http://cpc2.gao-lab.org/index.php) was used to predict the protein-coding ability of Lnc-LSG1. 22

CCK8 assay
The Cell Counting Kit-8 (Yeason, Hangzhou, China) was used to measured cell proliferation. In brief, 2 Â 10 3 ccRCC cells per well were seeded onto 96-well plates and 10 mL CCK-8 solution was added at days 1, 2, 3, and 4. After incubation at 37 C for 2 h, absorbance for each well was measured at 450 nm.

Transwell assay
Migration and invasion assays were conducted using 8-mm pore filters (Millipore, Germany) coated with or without Matrigel (BD Biosciences, San Jose, CA, USA). The ccRCC cells were seeded into the top chamber with serum-free RPMI 1640 or McCoy 5A, and 10% FBS medium was added to the lower chamber. After 24 h of incubation, non-migrating or non-invasive cells on the upper chamber were gently wiped off, and cells on the membrane bottom were fixed with 4% paraformaldehyde for 15 min. After fixation, cells were further stained with crystal violet for 20 min and counted in three randomly chosen fields.

Wound healing assay
The ccRCC cells were plated in 6-well plates and cultured to 90% confluency. The cell monolayer was scratched using a 1-mL pipette tip, washed twice with PBS, and cultured in 1% FBS medium. Images of wound closure in three random fields were captured after 0, 24, and 48 h incubation and further analyzed by Image J software.

IHC
All samples were paraffin embedded and cut into 4 mm sections. After deparaffinization, rehydration with alcohol, and antigen retrieval using sodium citrate buffer, the tissue sections were treated with 3% H 2 O 2 and blocked in 3% goat serum. Subsequently, the sections were incubated with METTL14 or ESRP2 antibodies at 4 C overnight, followed by incubation at room temperature for 1 h with secondary antibodies. The operator and the pathologists were blinded to the clinical and prognostic information of the patients.

RNA extraction and qRT-PCR
Total RNA was isolated with TRIzol (Cwbiotech, Peking, China) according to the manufacturer's instructions. A total of 1 mg RNA was used for cDNA synthesis by the All-in-One cDNA Synthesis Super-Mix for PCR (Bimake, Shanghai, China). The qRT-PCR reactions were performed using a 2Â SYBR Green qPCR master mix (Bimake). The primer sequences are listed in Table S5.

Western blotting
For this assay, ccRCC cells or human samples were lysed using RIPA buffer (Beyotime, Shanghai, China) for 15 min on ice and then centrifuged at 12,000 Â g for 20 min at 4 C. The supernatants were collected and added to 5Â loading buffer (Fdbio, Hangzhou, China). Proteins were further resolved in 10% SDS-PAGE, transferred onto a PVDF membrane (Bio-Rad, Hercules, CA, USA) and blocked in 5% nonfat milk. The membranes were immunoblotted with appropriate primary and secondary antibodies, and an enhanced chemiluminescence kit (Fdbio) was used to visualize specific protein bands.

RNA pull-down assay
The biotinylated probes were synthesized by Ribio (Guangzhou). Cells were lysed using IP buffer supplemented with RNase Inhibitor (Thermo Fisher Scientific, MA, USA) and protease inhibitor cocktail (Fdbio) at 4 C for 15 min. After centrifugation, the supernatants were collected and incubated with biotin-labeled RNA probes at 4 C overnight, followed by incubation with 20 mL streptavidin agarose beads for another 1 h at 4 C. Beads were collected and washed 10 times with IP buffer. Proteins retrieved by biotinylated RNA were analyzed by western blotting assay.

RNA immunoprecipitation assay
The RNA immunoprecipitation (RIP) experiments were performed using the Magna RIP RNA-Binding Protein Immunoprecipitation Kit (Millipore, MA, USA) according to the manufacturer's instructions. In brief, cells were lysed using RIP lysis buffer and incubated with the indicated antibodies and protein G magnetic beads, followed by protein digestion with proteinase K. The purified total RNA was subsequently subjected to qRT-PCR analysis.

MeRIP-seq assays
For MeRIP-seq, total RNA was isolated using TRIzol reagent. The obtained mRNA was further purified using the Dynabeads mRNA DIRECT Kit (Thermo Fisher) and fragmented by sonication. MeRIP-seq and library preparation were performed as per the reported protocol 33 with some modifications. In brief, sonicated mRNA was mixed with m 6 A antibody (Synaptic Systems, 202003) in IP buffer and incubated under head-to-tail mixing at 4 C for 2 h. The mixture was supplemented with protein A magnetic beads (Thermo Fisher) and incubated under head-to-tail mixing at 4 C for another 2 h. The beads were then washed with IP buffer three times before elution with m 6 A elution buffer two times. The eluates were combined and purified by an RNA Clean and Concentrator (Zymo, Orange, CA). The purified mRNA fragments were used to construct libraries with the TruSeq Stranded mRNA Library Prep Kit (Illumina, San Diego, CA). Sequencing was carried out on the Illumina HiSeq 2000 system with pair-end 150-bp read length. Reads were aligned to human genome version 38 (GRCh38) with TopHat. The longest isoform was retained if a gene had more than one isoform. Differential m 6 A-modified peaks between IP and input samples were identified using exomePeak (p < 0.01).

Measurement of total m 6 A mRNA levels
Total RNA was isolated using TRIzol reagent (Cwbiotech). The m 6 A level was measured using an m 6 A RNA methylation quantification kit (EpiGentek), according to the manufacturer's instructions.

FISH
The FISH probe labeled with Cy3 at the 5 0 of Lnc-LSG1 was purchased from RiboBio (Guangzhou, China). The subcellular localization of Lnc-LSG1 was further investigated using a FISH Kit (Ribobio) as recommended by the manufacturer. A confocal laser scanning microscope (Nikon, Tokyo, Japan) was used to capture the images.

Animal experiments
All animal studies were conducted in accordance with the institutional guidelines approved by the Animal Research Ethics Committee of Zhejiang University.
For the xenograft tumor model, approximately 1 Â 10 6 ccRCC cells suspended in 100 mL PBS were subcutaneously inoculated in the right flank of 5-week-old BALB/c nude mice. After 4 weeks, the xenograft tumors were collected and tumor volume was calculated according to the following formula: volume = (width 2 Â length)/2.
For the ccRCC orthotopic implantation model, approximately 1 Â 10 6 ccRCC cells suspended in 30 mL Matrigel were injected under the renal capsule of 5-week-old BALB/c nude mice. After 6 weeks, the anesthetized mice were intraperitoneally injected with D-luciferin (Yeason) and imaged using an in vivo imaging system to detect tumor growth and metastasis. The mice were then sacrificed, and the lung, liver, spleen, and intestine tissues were harvested, imaged, and subjected to IHC staining and H&E staining.
For the lung metastasis model, approximately 5 Â 10 5 ccRCC cells suspended in PBS were injected into the tail vein of 5-week-old mice. After 6-8 weeks, mice were anesthetized and lung metastasis was imaged as above. Lung tissues were further harvested, imaged, and subjected to H&E staining.

Statistical analysis
The statistical analysis of all experimental data was performed by GraphPad Prism 8.0. The survival curve of METTL14 was determined using the Kaplan-Meier method. All experiments were repeated more than three times. Statistical significance was considered as *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

AUTHOR CONTRIBUTIONS
G.L., L.X., D.S., and L.D. contributed to conceiving the project and designing the research. D.S., L.D., Z.L., R.W., C.Y., and H.W were responsible for the most of in vitro and in vivo experiments. X.W., W.X., H.Y., and L.X. performed part of in vitro experiments and data analysis. D.S., M.W., and S.Y. wrote the manuscript. S.Y. and S.Z. participated in data analysis. All authors have read and approved the final manuscript.